Extravillous trophoblast cell lineage development is associated with active remodeling of the chromatin landscape

The extravillous trophoblast cell lineage is a key feature of placentation and successful pregnancy. Knowledge of transcriptional regulation driving extravillous trophoblast cell development is limited. Here, we map the transcriptome and epigenome landscape as well as chromatin interactions of human trophoblast stem cells and their transition into extravillous trophoblast cells. We show that integrating chromatin accessibility, long-range chromatin interactions, transcriptomic, and transcription factor binding motif enrichment enables identification of transcription factors and regulatory mechanisms critical for extravillous trophoblast cell development. We elucidate functional roles for TFAP2C, SNAI1, and EPAS1 in the regulation of extravillous trophoblast cell development. EPAS1 is identified as an upstream regulator of key extravillous trophoblast cell transcription factors, including ASCL2 and SNAI1 and together with its target genes, is linked to pregnancy loss and birth weight. Collectively, we reveal activation of a dynamic regulatory network and provide a framework for understanding extravillous trophoblast cell specification in trophoblast cell lineage development and human placentation.


File Name: Supplementary Data 3b
Description: Chromatin accessibility in EVT cells. P values for called peaks estimated by a Poisson model followed by Benjamini and Hochberg procedure to adjust for multiple testing implemented in MACS3.

File Name: Supplementary Data 4a
Description: Differential binding affinity in chromatin accessibility regions in stem state cells. P values for called peaks and differential binding estimated by a Poisson model followed by Benjamini and Hochberg procedure to adjust for multiple testing implemented in MACS3 and DiffBind.

File Name: Supplementary Data 4b
Description: Differential binding affinity in chromatin accessibility regions in EVT cells. P values for called peaks and differential binding estimated by a Poisson model followed by Benjamini and Hochberg procedure to adjust for multiple testing implemented in MACS3 and DiffBind.

File Name: Supplementary Data 5
Description: Differential gene expression in EVT cells vs Stem state cells for genes mapping near (within 10kb) a chromatin accessibility region with differential binding and greater accessibility in EVT cells. P values for differential expression estimated by a Wald test followed by Benjamini and Hochberg procedure to adjust for multiple testing implemented in DESeq2. P values for called peaks and differential binding estimated by a Poisson model followed by Benjamini and Hochberg procedure to adjust for multiple testing implemented in MACS3 and DiffBind.

File Name: Supplementary Data 6
Description: 'Super-enhancers' overlapping a chromatin accessibility region with differential binding and greater accessibility in EVT cells. P values for called peaks and differential binding estimated by a Poisson model followed by Benjamini and Hochberg procedure to adjust for multiple testing implemented in MACS3 and DiffBind. P values for super-enhancers estimated by a Poisson model followed by Benjamini and Hochberg procedure to adjust for multiple testing implemented in HOMER.

File Name: Supplementary Data 7a
Description: Chromatin loops identified by Hi-C in stem state cells

File Name: Supplementary Data 8a
Description: Genes near (10kb) chromatin loop anchors where both anchors overlap a chromatin accessibility region with differential binding and greater accessibility in EVT cells. P values for differential expression estimated by a Wald test followed by Benjamini and Hochberg procedure to adjust for multiple testing implemented in DESeq2. P values for called peaks and differential binding estimated by a Poisson model followed by Benjamini and Hochberg procedure to adjust for multiple testing implemented in MACS3 and DiffBind.

File Name: Supplementary Data 8b
Description: Genes near (10kb) chromatin loop anchors where one anchor overlaps a chromatin accessibility region with differential binding and greater accessibility in EVT cells. P values for differential expression estimated by a Wald test followed by Benjamini and Hochberg procedure to adjust for multiple testing implemented in DESeq2. P values for called peaks and differential binding estimated by a Poisson model followed by Benjamini and Hochberg procedure to adjust for multiple testing implemented in MACS3 and DiffBind.

File Name: Supplementary Data 9a
Description: TF motif enrichment in chromatin accessibility region with differential binding and greater accessibility in EVT cells. P values estimated by a binomial model implemented in HOMER.

File Name: Supplementary Data 9b
Description: TF motif enrichment in differentially bound ATAC-Seq peaks overlapping H3K27ac mark in EVT cells. P values estimated by a binomial model implemented in HOMER.

File Name: Supplementary Data 9c
Description: TF motif enrichment in differentially bound ATAC-Seq peaks in EVT cells overlapping a 'super-enhancer'. P values estimated by a binomial model implemented in HOMER.

File Name: Supplementary Data 10
Description: Differential gene expression in EVT cells vs Stem state cells for transcription factor genes with motifs top ranked across all datasets. P values for differential expression estimated by a Wald test followed by Benjamini and Hochberg procedure to adjust for multiple testing implemented in DESeq2.

File Name: Supplementary Data 11
Description: Topmost differentially expressed EVT cell genes mapping near (10kb) chromatin loop anchors where one or both anchors overlap a chromatin accessibility region with differential binding and greater accessibility in EVT cells. P values for differential expression estimated by a Wald test followed by Benjamini and Hochberg procedure to adjust for multiple testing implemented in DESeq2.

File Name: Supplementary Data 12a
Description: Pathway analysis of top 500 most differentially expressed genes in EVT cells upon SNAI1 disruption. P values calculated using a right-tailed Fisher Exact Test implemented in Ingenuity Pathway analysis software.

File Name: Supplementary Data 12b
Description: Pathway analysis of top 500 most differentially expressed genes in EVT cells upon EPAS1 disruption. P values calculated using a right-tailed Fisher Exact Test implemented in Ingenuity Pathway analysis software.

File Name: Supplementary Data 13
Description: Differentially expressed genes in EVT cells upon EPAS1 disruption mapping to Cell Migration function.

File Name: Supplementary Data 14
Description: Chromatin accessibility regions with differential binding assessment in EVT cells overlapping EPAS1 binding motif and associated expression change of nearby gene upon EPAS1 disruption. P values for called peaks and differential binding estimated by a Poisson model followed by Benjamini and Hochberg procedure to adjust for multiple testing implemented in MACS3 and DiffBind. P values for differential expression estimated by a Wald test followed by Benjamini and Hochberg procedure to adjust for multiple testing implemented in DESeq2.

File Name: Supplementary Data 15
Description: Differentially expressed genes in EVT single-cells derived from idiopathic recurrent pregnancy loss vs normal control placentas. P values for differential expression estimated by a Wald test followed by Benjamini and Hochberg procedure to adjust for multiple testing implemented in DESeq2.

File Name: Supplementary Data 16
Description: Summary statistics from GWAS results in the EPAS1 gene region. P values estimated by a linear regression model.

File Name: Supplementary Data 17
Description: Expression of genes identified in GWAS of fetal genetic variants and birth weight following EPAS1 disruption in EVT cells. P values for GWAS estimated by a linear regression model. P values for differential expression estimated by a Wald test followed by Benjamini and Hochberg procedure to adjust for multiple testing implemented in DESeq2.